In collaboration with Dr. Ashlock, we have constructed a human artificial chromosome (HAC) vector from a 1 megabase yeast artificial chromosome (YAC). The YAC contained an alpha satellite DNA insert and was modified to have human telomeres and a putative human replication origin. The HAC vector was introduced into human cells in culture and HACs were identified which were mitotically stable in the absence of selection for more than 100 generations. The HACs were not derived from telomere fragmentation of endogenous chromosomes, but were larger than the original vector, suggesting the possibility of amplification and rearrangement. We are currently in the process of improving the design and maintenance of HAC vectors, using yeast genetic manipulation technologies. The yeast strains with humanized telomerase template, recently generated in our lab, will be used for the propagation of new HAC vectors. In addition, we are using the transformation-associated recombination (TAR) cloning technique in the yeast to clone large, unrearranged pieces of human chromosome centromeres. These new approaches and improved features will hopefully result in the production of more stable and reproducible HACs. - biotechnology research, gene therapy, genetics, human genome research,